Natural adaptation analysis is defined as analyzing the biochemical evolution of an organism to fit an ecological niche. At a molecular level, natural adaptation analysis illustrates how proteins evolve novel function by modifying existing structures. We are addressing the natural adaptation of the chymotrypsin family of serine proteases to cleave collagen. The degradation of collagen, the major structural protein in animals, is central to normal growth and development, as well as disease states such as arthritis and cancer. The molecular biology, biochemistry and structural biology of collagenolytic serine protease 1 from the fiddler crab, Uca pugilator, has been investigated. This enzyme possesses an unusually broad substrate specificity, encompassing those of trypsin, chymotrypsin, elastase and interstitial metallocollagenase. A unique arrangement of amino acids, including Gly 189 and Asp 226, in the primary binding pocket is thought to confer these specificities. Site-directed mutagenesis of trypsin was used to model the specificity determinants of crab collagenase. The resulting trypsin variants were characterized biochemically and crystallographically. A novel purification scheme was developed to isolate native collagenase from the fiddler crab hepatopancreas. The collagen specificity of the enzyme was determined and matched that for synthetic peptides. The primary collagen cleavage site is adjacent to that of the interstitial metallocollagenase. The crystal structure of crab collagenase was solved, providing the first view of an intact collagenolytic enzyme. Crab collagenase was cloned from a hepatopancreas cDNA library and expressed in S. cerevisiae. Quantitative structure-activity relationships determined that the collagenase active site is unique to the enzyme family. Site-directed mutagenesis of collagenase demonstrated that the primary substrate binding pocket has independent binding sites for basic and hydrophobic amino acids. A structural model for collagen recognition is presented. Modification of enzyme surface loops allows accessibility to and extended binding of the collagen triple helix. This natural adaptation analysis of crab collagenase will enable further studies to elucidate the molecular determinants of collagen recognition. This research was aided by the use of the Computer Graphics Lab.